POS-membrane contacts induce membrane deformation. (a–d) Confocal z-section analysis reveals that POS binding to the apical membrane of RPE, induces membrane protrusions rich in Dbl3, Cdc42-GTP, MRCKβ, and N-WASP. White dashes highlight the normal apical membrane. (e) Time course of POS incubation with primary RPE cells reveals that adhesion sites are enriched with receptor within 10 min of POS addition, as determined by adhesion receptor αvβ5 integrin staining and peak with respect to mean labeling intensity at 20 min. At 20 min, membrane deformation results in protrusions that transform into cups that wrap around POS at 30 min. Note, white arrowheads highlight POS-membrane adhesion sites and green arrowheads highlight cups. (f and g) Primary porcine RPE cells were treated with POS-FITC for 20 or 30 min and analyzed by confocal microscopy using colocalization software based on the Pearson’s correlation coefficient. Colocalization was calculated in grid three of the scatter charts. (h and i) Confocal xy scans of porcine primary RPE cells treated with POS for 30 min reveal cups encircling the particles with increased pMLC activity compared to POS-free segments of apical membrane. Treatment of cells with the MRCK inhibitor BDP5290 results in pseudopods negative for pMLC that are morphogenetically defective and unable to wrap around POS. White arrowheads highlight phagocytic cups. (j and k) Confocal xy scanning analysis of blebbistatin-treated primary porcine RPE cultures treated with POS-FITC for 1 h followed by 2 h chase. Cells were fixed and not permeabilized to detect external bound POS using anti-Annexin 5 antibody, in addition to total POS-FITC. Protein staining was quantified as mean intensity. Scale bars represent 10 μm. Analysis in k is based on n = 3 independent experiments and shows the data points, means ±1SD, the total number of cells analyzed for each type of sample across all experiments, and P values derived from t tests.